CN110699362B

CN110699362B - AFP5 gene and application thereof

Info

Publication number: CN110699362B
Application number: CN201911117789.8A
Authority: CN
Inventors: 马建忠; 邓子兵; 王永刚; 张伟杰; 蒲秀瑛
Original assignee: Lanzhou University of Technology
Current assignee: Lanzhou University of Technology
Priority date: 2019-11-15
Filing date: 2019-11-15
Publication date: 2023-03-24
Anticipated expiration: 2039-11-15
Also published as: CN110699362A

Abstract

The invention provides a sequence of a new gene AFP5 and application thereof, wherein an Arabidopsis thaliana cDNA library is screened by taking ABI5 protein as a bait, a new gene is cloned, and the sequence is named according to the characteristic of interaction between the gene and ABI 5: AFP5. Further, through the subcloning of AFP5 gene and the transformation of Arabidopsis, the applications of improving the seed germination rate of over-expression transgenic Arabidopsis plants, increasing the number of rosette leaves, widening the rosette leaves, increasing the plant height, flowering in advance of plants and the like are finally realized.

Description

AFP5 gene and application thereof

Technical Field

The invention relates to the field of plant genetic engineering, in particular to a novel gene (named AFP5 by the applicant) of arabidopsis thaliana and application thereof.

Background

The plant hormone abscisic acid (ABA) plays a major role in the initiation and maintenance of seed and shoot dormancy in plants, and in plant response to stress, particularly water stress. In addition, ABA affects many aspects of plant growth and development through interactions with other plant hormones.

ABI5 is the 5 th mutant found and defined in the ABA signaling pathway in plants, and is a basic leucine zipper type (bZIP) transcription factor. ABI5 interacting protein AFPs (ABI Five Binding Proteins) are a protein subfamily which is plant-specific and can be involved in ABA signal transduction and other plant growth and development process regulation. ABI5 is used as bait protein, a yeast two-hybrid system is utilized to screen an arabidopsis cDNA library, and a new gene AFP5 which can interact with ABI5 is cloned. Through amino acid residue sequence alignment analysis, the consistency of the AFP5 and the amino acid residue sequences of 4 arabidopsis AFPs found previously is respectively as follows: 23.19% identity with AFP1, 24.07% identity with AFP2, 25.00% identity with AFP3, and 21.25% identity with AFP 4. Although the overall identity of amino acid residue sequences of subfamilies of AFPs is not high, three highly conserved regions A, B and C still exist for the amino acid residue sequences of members of this subfamily. The function of both a and C is unknown, except for the presence of the putative nuclear localization sequence in conserved region B. Our structural analysis work showed that conserved region C of AFPs alone appears to have a stronger effect with ABI5. There are also weaker interactions between 5 AFPs, but the physiological significance of this interaction is currently unknown. Compared with wild arabidopsis thaliana plants (Columbia ecotype), the arabidopsis thaliana plants (OE 2, OE4 and OE 5) over-expressing the AFP5 gene show genetic phenotypes of improved seed germination rate, early flowering, higher plant type and the like.

The problems existing in the prior art are as follows: (1) The gene sequence of AFP5 (ABI Five Binding Protein 5), the 5 th interacting Protein of ABI5, has not been reported. (2) the biological function of AFP5 has not been reported in prior studies.

Disclosure of Invention

The key technical problem to be solved by the invention is to provide a new gene AFP5 sequence and application thereof, and finally the applications of increasing the seed germination rate, increasing the number of rosette leaves, widening the rosette leaves, increasing the plant height, flowering in advance and the like of an over-expressed transgenic arabidopsis plant are realized by cloning AFP5 and converting arabidopsis.

In order to solve the technical problems, the invention adopts the following technical scheme:

1. an arabidopsis thaliana new gene AFP5, the CDS sequence of which is shown in No. 1; the full-length cDNA sequence is shown in No. 2.

2. An arabidopsis thaliana new gene AFP5 cloning method comprises the following steps: (1) total RNA extraction reverse transcription; (2) full-length amplification of AFP 5; and (3) AFP5 yeast two-hybrid vector construction and sequence determination.

3. The AFP5 interacting protein screening and gene name determining method includes: (1) preparing yeast competent cells; (2) transforming the recombinant plasmid into a yeast competent cell; and (3) verifying the interaction between AFP5 and ABI5.

4. An AFP5 overexpression transgenic Arabidopsis plant genetic transformation method, comprising:

(1) Constructing an AFP5 overexpression vector; (2) transforming agrobacterium GV3101 by an AFP5 overexpression vector; (3) transforming arabidopsis thaliana by an agrobacterium tumefaciens floral dip method; (4) And (4) screening and identifying AFP5 overexpression transgenic arabidopsis thaliana plants.

5. Application of AFP5 in improving germination rate of Arabidopsis seeds.

6. Use of AFP5 for increasing the number of rosette leaves of young plants.

7. Use of AFP5 in advanced flowering phase.

8. Use of AFP5 for increasing plant height.

9. Application of AFP5 in increasing sensitivity of plants to ABA.

Drawings

FIG. 1 is an alignment analysis of the sequences of the AFPs family members of the present invention.

Wherein, the comparison and analysis of the amino acid sequences of AFPs families by using DNAman software (above) shows that I, II, III and IV are four conserved regions. (Next) phylogenetic tree analysis. AFP1 and AFP2 belong to the same clade, and AFP4 and AFP5 belong to the same clade.

FIG. 2 is a schematic representation of the yeast two-hybrid system of the present invention demonstrating the interaction of AFP5 with ABI5.

Wherein, 1 is BD + AD,2 is BD + AD-AFP 5+ AD,3 is BD + AD-ABI5,4 is BD-AFP5+ AD-ABI5.

FIG. 3 shows the result of RT-PCR identification after AFP5 overexpression transgenic Arabidopsis seedlings of the invention grow for 7 d.

FIG. 4 is a graph showing that AFP5 overexpressing transgenic Arabidopsis lines of the invention exhibit a high germination phenotype.

Wherein (above) seed germination after 7 days of growth on 1/2MS medium (16 hours light, 8 hours dark). And (lower) counting the germination rate of the seeds on a 1/2MS culture medium. (n =30-60, error bars ± SE,16h light and 8h dark).

FIG. 5 shows that AFP5 overexpressing transgenic Arabidopsis lines of the invention show a lotus throne leaf broadening phenotype.

FIG.6 is a phenotype of AFP5 overexpressing transgenic Arabidopsis lines of the invention showing an increased number of rosette leaves.

FIG.7 is the early flowering phenotype of an AFP5 overexpressing transgenic Arabidopsis line of the invention.

FIG.8 is a more ABA sensitive phenotype of seed germination of an AFP5 overexpressing transgenic Arabidopsis line of the invention.

Detailed description of the invention

The methods and devices used in the following examples of the present invention are conventional methods and devices unless otherwise specified; the equipment and the reagent are all conventional equipment and reagents purchased by a reagent company. In order to make the objects, technical solutions and advantages of the present invention more apparent, the following detailed description of the embodiments of the present invention is provided in connection with the specific embodiments. Examples of these preferred embodiments are illustrated in the specific examples.

It should be noted that, in order to avoid obscuring the technical solutions of the present invention with unnecessary details, only the technical solutions and/or processing steps closely related to the technical solutions of the present invention are shown in the embodiments, and other details that are not relevant are omitted.

Example 1

This example provides a method for cloning AFP5 gene and constructing a vector, comprising:

(1) Total RNA extraction and reverse transcription

Total RNA extraction was performed on the entire Columbia ecotype Arabidopsis thaliana (Arabidopsis thaliana Columbia) grown on 1/2MS solid medium for about one week according to the general RNA extraction kit (Tiangen, DP 437). The extracted RNA was subjected to reverse transcription in the following order after the concentration of RNA was determined using NanoDrop 2000:

mix the following on ice:

after 5min of reaction at 65 ℃, quickly taking out the mixture for ice bath for 10min, adding the following substances into the tube, and gently mixing the substances uniformly:

after 60min at 37 ℃ the reaction was stopped by leaving it at 70 ℃ for 15 min. After completion of reverse transcription, ddH was added ₂ The cDNA concentration was diluted to 20 ng/. Mu.l O.

(2) full-Length amplification of AFP5

Designing a specific primer pair according to the sequence of the AFP5 gene, and carrying out PCR amplification by using the cDNA template obtained in the step (1). The PCR system is as follows:

the amplification parameters of the PCR were: pre-denaturation at 95 ℃ for 5min,30 cycles comprising denaturation at 95 ℃ for 15s, annealing at 57 ℃ for 30s, extension at 72 ℃ for 1min, and finally extension at 72 ℃ for 10min. After the PCR was completed, agarose gel electrophoresis was performed.

(3) AFP5 yeast two-hybrid vector construction and sequence determination

Based on the full-length cDNA sequence encoding AFP5, a pair of specific amplification primers was designed as follows:

F1:5’-CCGGAATTCATGTTTGTTGAATCTTTATCAAT-3’；

R1:5’-AACTGCAGTCACAAAGTTGAGGGAAC-3’。

restriction enzyme sites (underlined) EcoR I and Pst I are respectively introduced into the 5' ends of the upstream primer and the downstream primer, and 2-3 protective bases are added. The PCR product amplified by a specific primer pair with cDNA as a template is recovered and is subjected to double enzyme digestion with restriction enzymes EcoR I and Pst I respectively with a vector pGBKT7, the PCR product is connected by T4 DNA ligase and transformed into an escherichia coli E.coli DH5 alpha competent cell, 6 single colonies are selected for colony PCR identification and double enzyme digestion identification, then the obtained product is sent to Suzhou Jinzhi biotechnology limited for sequencing, and the recombinant plasmid with correct sequencing is named as pGBKT7-AFP5.

Example 2

This example provides for the selection of AFP5 interacting proteins and determination of gene names, including:

(1) Preparation of Yeast competent cells

Selecting yeast AH109 single colony with age of about 2 weeks and diameter of about 3mm from YPDA solid medium surface, inoculating into 0.5ml YPDA liquid medium, vortex shaking for about 3min to disperse the collected yeast cells, inoculating into 10ml YPDA liquid medium, and shake culturing at 30 deg.C and 250r/min for about 18 hr until thallus OD ₆₀₀ The value reaches more than 1.5. The overnight culture was inoculated into 250ml YPDA liquid medium and the initial microbial cells OD were allowed to stand ₆₀₀ Culturing at 30 deg.C and 250r/min for 4 hr until the thallus OD ₆₀₀ The value is between 0.4 and 0.6. Centrifuging at 1000 Xg and room temperature for 5min, carefully pouring off the supernatant, adding 20ml of 1 XTE/LiAc solution to resuspend the yeast cells, centrifuging at 1000g and room temperature for 5min, carefully discarding the supernatant, and repeating the steps. About 3ml of 1 XTE/LiAc solution was added to the precipitated yeast cells and the cells were gently suspended, i.e., yeast competent cells. The competent cells can be kept in a refrigerator or ice bath at 4 ℃ for a period of time not exceeding 4 hours.

(2) Recombinant plasmid transformed yeast competent cell

Respectively sucking the recombinant plasmids to be transformed and placing the recombinant plasmids into a 1.5ml sterile centrifuge tube according to Table 1 for mixing, then adding 10 mu l of salmon Sperm DNA (term DNA,10 mg/ml), repeatedly blowing and uniformly mixing by a pipette, slowly reversing and uniformly mixing the yeast competent cells, respectively sucking 100 mu l of the yeast competent cells and adding the yeast competent cells into the centrifuge tube, and slowly blowing and uniformly mixing by the pipette. Adding 600 μ l of 1 XPEG/LiAc into each centrifuge tube, mixing by vortexing, shaking at 30 deg.C and 150rpm for 40min, adding 70 μ l of 100% DMSO each, mixing by slow inversion, placing in 42 deg.C water bath for 15min, and rapidly ice-cooling for 2min. Centrifuging at 12000rpm for 30s at room temperature, slightly sucking and removing the supernatant, adding 500 mu l of 1 XTE, blowing and uniformly mixing, sucking 100 mu l of suspension, uniformly coating the suspension on the surface of the SD/-LW auxotrophic solid culture medium, and after the liquid on the surface is volatilized, inversely culturing for 4-6 days in a constant-temperature incubator at 30 ℃ until the diameter of the grown bacterial colony is about 1-2 mm.

Table I yeast two-hybrid recombinant vector co-transformation combination

(3) Demonstration of AFP5 and ABI5 interaction

Single colonies in the several steps (2) were picked up in 3ml of fresh SD-LW liquid medium and cultured overnight at 30 ℃ and 220 rpm. The colony growth was observed by pipetting 1. Mu.l of the culture spot in solid medium SD-LW, SD-AHLW, X- α -Gal + SD-AHLW, and culturing in an inverted state in a 30 ℃ incubator for 2-3 days as shown in FIG. 2.

The results show that: AFP5 can interact with ABI5.

Example 3

This example provides a method for genetic transformation of an AFP5 overexpressing transgenic arabidopsis plant comprising:

(1) AFP5 overexpression vector construction

F2:5’-GGACTAGTATGTTTGTTGAATCTTTATCAAT-3’；

R2:5’-GGACTAGTACTCAAAGTTGAGGGAAC-3’。

the 5' ends of the upstream primer and the downstream primer are respectively introduced with a restriction enzyme site (underlined) Spe I, and 2-3 protective bases are added. PCR products amplified by a specific primer pair by taking pGBKT7-AFP5 plasmid as a template are recovered and then are respectively cut by restriction enzyme Spe I (the pCAMBIA1304 is subjected to dephosphorylation treatment after being cut by Spe I), the PCR products are connected and transformed into escherichia coli E.coli DH5 alpha competent cells by T4 DNA ligase, 6 single colonies are selected for colony PCR identification and enzyme digestion identification and then are sent to Suzhou Jinzhi Zhi Biotech limited for sequencing, and the plasmid with correct sequencing is named as pCAMBIA1304-AFP5.

(2) AFP5 overexpression vector transformation agrobacterium GV3101

Mu.l of recombinant plasmid pCAMBIA1304-AFP5 was taken and mixed uniformly in 1 Agrobacterium tumefaciens GV3101 competent cells. Standing on ice for 30min, quickly freezing with liquid nitrogen for 1min, thermally shocking at 42 deg.C for 90s, and quickly freezing for 2min. 800. Mu.l of fresh LB liquid medium was added and shake-cultured at 28 ℃ for 4 hours. Mu.l of the suspension was spread on a resistant LB plate (containing Gen 40. Mu.g/ml, rif 20. Mu.g/ml, kan 40. Mu.g/ml) and cultured in 28 ℃ inverted culture for 2-3 days. 3 single colonies were picked up in 3mL of fresh LB (Gen 40. Mu.g/mL, rif 20. Mu.g/mL, kan 40. Mu.g/mL) liquid medium and cultured overnight at 220rpm at 28 ℃.1 mul of bacterial liquid is taken respectively for colony PCR identification. The colony PCR positive transformant further genetically transforms Arabidopsis thaliana.

(3) Agrobacterium flower-soaking method for transforming arabidopsis

a. Before transformation, columbia ecotype Arabidopsis plants growing for about 25 days are prepared, after the Arabidopsis plants begin to sprout, the top end of the main inflorescence of the Arabidopsis is cut off, and the axillary inflorescence is prevented from being damaged, so as to induce the generation of the lateral inflorescence. The transformation is carried out when the side branches extend 2-10 cm. And watering the Arabidopsis plants needing to be infected based on the condition that more unopened buds exist.

b. Agrobacterium containing AFP5 overexpression vector pCAMBIA1304-AFP5 was inoculated into 5ml of fresh LB (containing Gen 40. Mu.g/ml, rif 20. Mu.g/ml, kan 40. Mu.g/ml) liquid medium, and shake-cultured at 30 ℃ and 200r/min for about 15 hours in a shaker.

c. The culture in the previous step is transferred into 200ml of fresh LB liquid culture medium containing corresponding antibiotics according to the proportion of 1 ₆₀₀ ＝0.8-1.0。

d. Centrifuging at 4000r/min for 15min to collect thallus, and regulating solution OD with permeation buffer (1/2 MS culture medium +5% sucrose) ₆₀₀ ＝0.8-1.0, and adding Silwett L-77 (SINOPCR S5605) with final concentration of 0.03% and mixing.

e. The pollinated flowers and fruit pods were removed with scissors and the plants were watered one day ahead with enough water. Each flower of the prepared Arabidopsis thaliana plants was stained by pipetting the Agrobacterium suspensions containing the four plasmids to be transformed, respectively, with a 5ml syringe. The impregnated plants (T0 generation plants) were covered with a freshness wrap to maintain humidity.

f. Placing the soaked Arabidopsis thaliana plant in the dark for culturing for 12h, then placing the plant back to normal conditions (22 ℃,16h light and 8h dark) for culturing, removing the freshness protection package after 2 days, continuously culturing until the pod is mature, and repeatedly soaking for 2-3 times in the period to improve the transformation efficiency.

g. When the plant grows for 2-3 weeks, the pod at the top end turns yellow, arabidopsis thaliana seeds (T1 generation) are collected according to the number before the pod cracks, and are stored after being fully dried so as to screen transgenic arabidopsis thaliana plants.

(4) Screening and identification of AFP5 overexpression transgenic arabidopsis plants

Adding a small amount of sterile water to immerse the T1 generation seeds, and vernalizing in a refrigerator at 4 deg.C for 3-4 days. Sterilizing in a clean bench, spreading on 1/2MS solid culture medium containing 25 μ g/ml hygromycin, and culturing in plant light incubator (22 deg.C, 16h light, 8h dark, humidity of about 60%, and light intensity of 3000-5000 Lx). After two weeks of growth, the suspected positive transgenic Arabidopsis plants can grow normally, while the non-transgenic plants show yellow cotyledons and short roots and cannot grow into the culture medium. The positive transgenic arabidopsis thaliana plants are transplanted into nutrient soil (Denmark substrate No. 5, pH 5.5) for culture (22 ℃,16h of light, 8h of darkness, humidity of about 60 percent and illumination intensity of 3000-5000 Lx), and mature seeds (T2 generation seeds) are harvested in a single plant. The T2 generation seeds were further screened on MS medium containing 25. Mu.g/ml hygromycin resistance to T3 generation, followed by screening to T4 generation for phenotypic analysis.

Specific primers (AFP 5RTF and AFP5 RTR) are designed according to the sequence of pCAMBIA1304-AFP5 recombinant plasmid, and the expression condition of the AFP5 gene in an antibiotic positive Arabidopsis plant is identified by RT-PCR. Total RNA is extracted from 7-day-old wild-type and AFP5 overexpression transgenic Arabidopsis plants (cultured on a 1/2MS plate), reverse-transcribed into cDNA, and amplified by specific primers AFP5RTF and AFP5 RTR.

AFP5RTF:5’-AFP5RTF:AGCTCTATCAACATGTGTAACGG-3’；

AFP5RTR:5’-AGAAGAGCTGCTAGGATCGG-3’

The expression of the overexpressed genes in transgenic Arabidopsis was analyzed by RT-PCR. The RT-PCR electrophoresis result shows specificity bands with corresponding molecular weight, and the bands of over-expression strains (OE 2, OE4 and OE 5) are more obvious than the bands of wild type (Col), as shown in figure 3.

The results show that: AFP5 was successfully embedded in the Arabidopsis genome and was capable of overexpression.

Example 4

This example provides an application of AFP5 in improving germination rate of arabidopsis thaliana seeds, including:

harvested mature T4 generation seeds and wild type seeds were added to ddH2O and placed in a refrigerator at 4 ℃ for vernalization for 3-4 days. After being sterilized in a clean bench, the culture medium is spread on the same 1/2MS solid culture medium containing 25 mu g/ml hygromycin and is placed in a plant illumination incubator for culture (22 ℃,16h of illumination, 8h of darkness, humidity of about 60 percent and illumination intensity of 3000-5000 Lx). And (5) counting the germination rate of the seeds of 1-7 days.

After 7d, the germination rate of AFP5 overexpression transgenic Arabidopsis strains (OE 2, OE4 and OE 5) is obviously higher than that of wild Arabidopsis plants, as shown in figure 4.

The results show that: the germination rate of arabidopsis seeds can be obviously improved by over-expressing the AFP5 gene.

Example 5

This example provides the use of AFP5 to alter the leaf width of a seedling rosette, comprising:

a. all plants are selected from flowerpots with the same specification and square calibers, certain nutrient soil (No. 5 Danish matrix, pH 5.5) is put into each flowerpot, the flowerpots are placed in the same flat-bottomed tray, and water is added into the tray so that the soil in the flowerpots in the tray can absorb water automatically.

b. After purifying AFP5 overexpression transgenic arabidopsis thaliana strains (OE 2, OE4 and OE 5) T4 generation seeds and wild type seeds, dibbling the seeds in nutrient soil (Denmark substrate No. 5, pH 5.5), and then placing the seeds in a greenhouse for culture (22 ℃,16h of light, 8h of dark, about 60 percent of humidity and 3000-5000 Lx of light intensity). And removing the seedlings with poor growth conditions and redundant seedlings after one week, so that 5 seedlings with more consistent growth conditions are reserved in each flowerpot.

c. Observing and counting the growth condition of the lotus throne leaves of Arabidopsis after 3 weeks of growth

AFP5 overexpression transgenic Arabidopsis lines (OE 2, OE4 and OE 5) rosette leaves all showed a broader phenotype than wild type lines rosette leaves as shown in FIG. 5.

The results show that: the AFP5 gene is overexpressed, so that the width of the leaf blade of the lotus throne of an arabidopsis plant can be obviously increased.

Example 6

This example provides the use of AFP5 to increase the number of rosette leaves in seedlings, comprising:

b. Placing AFP5 overexpression transgenic Arabidopsis strains (OE 2, OE4 and OE 5) T4 generation seeds and wild type seeds in 4 ℃ for vernalization for 3-4 days, dibbling the seeds in nutrient soil (Denmark substrate No. 5, pH 5.5), and placing the seeds in a greenhouse for culture (22 ℃,16h of light, 8h of darkness, humidity of about 60 percent and light intensity of 3000-5000 Lx). After one week, the seedlings with poor growth conditions and redundant seedlings are removed, and 5 seedlings with similar growth conditions are reserved in each flowerpot.

AFP5 overexpressing transgenic Arabidopsis lines (OE 2, OE4 and OE 5) rosette leaves all showed more phenotype than wild type lines (FIG. 6).

The results show that: the overexpression of the AFP5 gene can obviously increase the number of leaves of a lotus throne of an arabidopsis plant.

Example 7

This example provides the use of AFP5 in advanced flowering phase, comprising:

a. all plants are selected from flowerpots with the same specification and square calibers, the same nutrient soil (No. 5 Danish substrate, pH 5.5) is put into each flowerpot, the flowerpots are placed in the same flat-bottomed tray, and water is added into the tray so that the soil in the flowerpots in the tray can automatically absorb water.

b. AFP5 overexpression transgenic arabidopsis strains (OE 2, OE4 and OE 5) T4 generation seeds and wild type arabidopsis seeds are placed at 4 ℃ for vernalization for 3-4 days, dibbled in nutrient soil (Denmark substrate No. 5, pH 5.5) and then placed in a greenhouse for culture (22 ℃,16h of illumination, 8h of darkness, humidity of about 60 percent and illumination intensity of 3000-5000 Lx). After one week, the seedlings with poor growth conditions and redundant seedlings are removed, and 5 seedlings with similar growth conditions are reserved in each flowerpot.

c. Observing and counting the flowering time and growth condition of arabidopsis

AFP5 overexpressing transgenic Arabidopsis lines (OE 2, OE4 and OE 5) flowered 4 weeks later. Where OE4 blooms earliest, OE2 times later, and OE5 slightly later than the first two lines. However, all over-expression lines flowering earlier than the wild-type line (FIG. 7)

The results show that: the flowering time of Arabidopsis plants overexpressing the AFP5 gene is significantly advanced.

Example 8

This example provides the use of AFP5 to increase plant height, comprising:

b. AFP5 overexpression transgenic Arabidopsis strains (OE 2, OE4 and OE 5) T4 generation seeds and wild type Arabidopsis seeds are placed at 4 ℃ for vernalization for 3-4 days, dibbled on nutrient soil (Denmark Denshoku's matrix No. 5, pH 5.5), and then placed in a greenhouse for culture (22 ℃,16h of light, 8h of dark, about 60% of humidity and 3000-5000 Lx of light intensity). After one week, the seedlings with poor growth conditions and redundant seedlings are removed, and 5 seedlings with similar growth conditions are reserved in each flowerpot.

c. Observing and counting the height of Arabidopsis plants

AFP5 overexpressing transgenic Arabidopsis lines (OE 2, OE4 and OE 5) entered the end of the growth cycle at 8 weeks. In this case, the transgenic line OE2 had an average plant height of 37cm, OE4 had an average plant height of 38cm, OE5 had an average plant height of 34cm, and the wild-type plant height was 32cm (FIG. 7).

The results show that: AFP5 gene overexpression significantly increases plant height.

Example 9

This example provides the use of AFP5 to increase the sensitivity of plants to ABA, comprising:

AFP5 overexpression transgenic line T4 generation seeds and wild type Arabidopsis seeds are added into ddH ₂ And O, vernalizing for 3-4 days in a refrigerator at 4 ℃. After disinfection in a clean bench, the plants are dibbled on 1/2MS plates with different ABA contents (the ABA concentrations are 0.1 MuM, 0.5 MuM and 1 MuM respectively), and the plants are cultured in a plant illumination incubator (22 ℃,16h of illumination, 8h of darkness, about 60 percent of humidity and 3000-5000 Lx of illumination intensity). And (5) counting the germination rate of the seeds of 1-7 days.

After 7d, the germination rate of AFP5 overexpression transgenic Arabidopsis strains (OE 2, OE4 and OE 5) is obviously lower than that of wild type Arabidopsis plants (FIG. 8), thereby showing that the seed germination of the AFP5 overexpression transgenic Arabidopsis strains is more sensitive to ABA than that of the wild type plants.

The results show that: the sensitivity of the plant to ABA can be obviously enhanced by over-expressing the AFP5 gene.

Has the advantages that:

the invention has at least the following beneficial effects: 1. the applicant screened a yeast two-hybrid library of arabidopsis cDNA using ABI5 gene as bait, cloned a new gene, and named AFP5 based on its interactivity with ABI 5.2. Analysis by alignment with previously reported amino acid residue sequences of 4 AFPs subfamily members showed that the AFP5 protein also contains three conserved regions in common: A. b and C. However, the overall identity of the amino acid residue sequences of the AFP5 protein to other AFPs proteins is not high: 23.19% identity with AFP1, 24.07% identity with AFP2, 25.00% identity with AFP3, and 21.25% identity with AFP 4. 3. The seed germination rate of an Arabidopsis plant over-expressing AFP5 gene is improved, the rosette leaf is widened, the number of rosette leaf is increased, the plant blooms in advance, and the plant height is increased. 4. The CDS nucleotide residue sequence of the cloned AFP5 gene is: sequence 1.5. The full-length cDNA nucleotide residue sequence of the cloned AFP5 gene is: sequence 2.

The foregoing is directed to embodiments of the present application and it is noted that numerous modifications and adaptations may be made by those skilled in the art without departing from the principles of the present application and are intended to be within the scope of the present application.

<110> Lanzhou university of science and engineering

<120> AFP5 gene and use thereof

<160> 2

<210> 1

<211> 882

<212> DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 1

1 ATGTTTGTTG AATCTTTATC AATGGCAGAA AAGTTTGTAG AAGATGAGAA TGTGGAGGTC

61 GAGCTCGAGC TCGAGCTATG TCTTTCCCTG GGAGGTCCTT TCAAGAAAAC GGAGAAATCT

121 AAAACATTTG GACAATCTAA CGCCGTCGGA TTCGAGAAGG ATAACGGCGT TAATCTTGAC

181 GGCAGAACGA CGAATGTGAC GAGGATAAAG GAAACACGGA AGAAGCGGGA GGCGAAGCAG

241 CAGCAGAGAA GCGGAGAAGA AGGAGAGTGC AAGAGGATCA GAACAGAATA TAACGGAGTT

301 AGTAACGGCG ATGATATGGA TTTGAGCTCT ATCAACATGT GTAACGGTTA CGGGTCGGGT

361 CGACTCAAGG AAAGCAGTAA AGACGTTACG ATTGGGTCAC CCATTTGCAC CTCCTCCTCC

421 GTATCCGATC CTAGCAGCTC TTCTCGCCAA GAAGGTGGAA GTGGCGACAT TGGGGCACAA

481 TCTGGCCAAA CCAAACAGGT TAGATCTCCG GTTAACAACA TTCTGACCGG TACAGAGCAA

541 ACCGTGCATA ACACAGACGG TTCAAAAGAC GCGGAGACCC AAGAATGGTC CAACTCTTCT

601 GTAACCAAAG AGAGTGGGAA GCCGCCAAAA CCGCATACCA ACAGTAACGG TAACGGCAGT

661 TTGCTTCCGT TTGCTCAGAT GCCTTGCGTG ACTAGCACCG GTAACGGTCC CGAGGGCAAA

721 ACCGTAAATG GTTTTCTATA CCGTTACTCA AAATCAGAGA TCAGTATAAT CTGCGTCTGC

781 CATGGAACGT CTTTCTCGCC GGCTGAGTTC ATCGTTCACG CCGGTGGGAC TAATGTGTCG

841 CATCCGTTGA GGCATATAAC CGTTGTTCCC TCAACTTTGT GA

<210> 1

<211> 1099

<212> DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 2

1 CGAAGCTTCA AAACCCTTCA AGAGGAAAAT TTTGCGGAAG ACAAGAGACC ATCTTTTTGA

61 CATTATATGT TTGTTGAATC TTTATCAATG GCAGAAAAGT TTGTAGAAGA TGAGAATGTG

121 GAGGTCGAGC TCGAGCTCGA GCTATGTCTT TCCCTGGGAG GTCCTTTCAA GAAAACGGAG

181 AAATCTAAAA CATTTGGACA ATCTAACGCC GTCGGATTCG AGAAGGATAA CGGCGTTAAT

241 CTTGACGGCA GAACGACGAA TGTGACGAGG ATAAAGGAAA CACGGAAGAA GCGGGAGGCG

301 AAGCAGCAGC AGAGAAGCGG AGAAGAAGGA GAGTGCAAGA GGATCAGAAC AGAATATAAC

361 GGAGTTAGTA ACGGCGATGA TATGGATTTG AGCTCTATCA ACATGTGTAA CGGTTACGGG

421 TCGGGTCGAC TCAAGGAAAG CAGTAAAGAC GTTACGATTG GGTCACCCAT TTGCACCTCC

481 TCCTCCGTAT CCGATCCTAG CAGCTCTTCT CGCCAAGAAG GTGGAAGTGG CGACATTGGG

541 GCACAATCTG GCCAAACCAA ACAGGTTAGA TCTCCGGTTA ACAACATTCT GACCGGTACA

601 GAGCAAACCG TGCATAACAC AGACGGTTCA AAAGACGCGG AGACCCAAGA ATGGTCCAAC

661 TCTTCTGTAA CCAAAGAGAG TGGGAAGCCG CCAAAACCGC ATACCAACAG TAACGGTAAC

721 GGCAGTTTGC TTCCGTTTGC TCAGATGCCT TGCGTGACTA GCACCGGTAA CGGTCCCGAG

781 GGCAAAACCG TAAATGGTTT TCTATACCGT TACTCAAAAT CAGAGATCAG TATAATCTGC

841 GTCTGCCATG GAACGTCTTT CTCGCCGGCT GAGTTCATCG TTCACGCCGG TGGGACTAAT

901 GTGTCGCATC CGTTGAGGCA TATAACCGTT GTTCCCTCAA CTTTGTGAAC TACAAATTTA

961 CATATTTTGT TACTCTTTTT GTCTCGTTTT TGTGTATATA AGTTTCACCG ATGGACGTTT

1021 TGAGTTCGAA GGGACTCTCC ATCCATGATT TTCTTAGAGT TTTGTTTTTC CTTGACATTT

1081 TTCAACTAAT TTTACACTT

Claims

The application of AFP5 in improving the germination rate of arabidopsis seeds is characterized in that the application is realized by over-expressing an AFP5 gene, and the CDS sequence of the AFP5 is shown as SEQ ID NO:1 is shown in the specification; the full-length cDNA sequence is shown as SEQ ID NO:2, respectively.
The application of AFP5 in increasing the number of rosette leaves of arabidopsis seedlings is characterized in that the application is realized by over-expressing an AFP5 gene, and the CDS sequence of the AFP5 is shown as SEQ ID NO:1 is shown in the specification; the full-length cDNA sequence is shown as SEQ ID NO:2, respectively.
The application of AFP5 in advancing arabidopsis flowering phase is characterized in that the application is realized by over-expressing an AFP5 gene, and a CDS sequence of the AFP5 is shown as SEQ ID NO:1 is shown in the specification; the full-length cDNA sequence is shown as SEQ ID NO:2, respectively.
The application of AFP5 in increasing the height of an arabidopsis thaliana plant is characterized in that the application is realized by over-expressing an AFP5 gene, and the CDS sequence of the AFP5 is shown as SEQ ID NO:1 is shown in the specification; the full-length cDNA sequence is shown as SEQ ID NO:2, respectively.
The application of AFP5 in increasing the sensitivity of arabidopsis thaliana plants to abscisic acid is characterized in that the application is realized by over-expressing an AFP5 gene, and the CDS sequence of the AFP5 is shown as SEQ ID NO:1 is shown in the specification; the full-length cDNA sequence is shown as SEQ ID NO:2, respectively.